Application Of Animal Models In Diabetic Cardiomyopathy

Lee WS, et al.showed normal cardiac function in the unstressed state but developed maladaptive hypertrophy and severe contractile dysfunction in response to left ventricular (LV) pressure overload[13,25]. Therefore, GLUT4 is required for the maintenance ofcardiac function and structure in response to pathological processes that increase energy demand, in part through secondarychanges in mitochondrial metabolism and cellular stress survival signaling, such as the phosphoinositide 3-kinase (PI3K)–Akt pathway [13,25].In addition to stimulating glucose uptake, both insulin signaling [13,26] and cardiomyocyte contraction [13,27] can promote fatty acid uptake into cardiomyocytes via induction ofcluster of differentiation 36 (CD36) translocation to sarcolemma membranes [26,28]. The long-lasting presence of CD36 atthe sarcolemma membrane leads to an increased rate of longchain fatty acid uptake and accumulation of triglycerides incardiomyocytes, which results in lipotoxic DiaCM [28,29].The transcription factor peroxisome proliferator-activatedreceptor-α (PPARα) is a major regulator of lipid metabolismand can increase the expression of genes encoding CD36, fattyacid-binding proteins and proteins involved in β-oxidation inthe mitochondria and peroxisome [13,30]. Tribbles-relatedprotein 3 (TRB3) can directly bind to Akt and inhibit Aktphosphorylation [13,31,32]. The expression of TRB3 is upregulated in the heart in T1DM and T2DM rodent models [33,34]and in skeletal muscle in patients with T2DM [32]. Furthermore, a rat model of T2DM induced by a HFD and low-dosestreptozotocin (STZ) demonstrated severe insulin resistanceand properties of DiaCM, including myocardial fibrosis, cardiac inflammation and LV dysfunction, in addition to increasedexpression of TRB3, compared with control rats [34].The hearts from rats with T2DM infused ex vivo with theCD36 inhibitor sulfo-N-succinimidyl oleate (SSO) before inducing hypoxia, which resulted in a 29% reduction in the rateof fatty acid oxidation and an approximately 50% reduction intriglyceride concentration compared with vehicle treatment,showed a restoration of fatty acid metabolism to control levelsfollowing hypoxia–reoxygenation [13,35]. SSO infusion intodiabetic rat hearts ex vivo before hypoxia also prevented cardiac dysfunction [35]. Fenofibrate treatment prevented fibrosisand diastolic dysfunction in diabetic rats, probably throughimprovements in cardiac and systemic lipid metabolism[36,37]. Fenofibrate treatment was also associated with reductions in markers of apoptosis and cardiac hypertrophy in ratswith STZ-induced T1DM [38]. The glucagon-like peptide-1132(GLP1) analog liraglutide protected against the developmentof DiaCM in a rat model of STZ-induced T1DM by inhibitingthe endoplasmic reticulum (ER) stress pathway [39]. Similarly,the GLP1 analog exendin-4 prevented the development of DiaCM via the amelioration of lipotoxicity in a mouse model ofT2DM [40]. The dipeptidyl peptidase-4 (DPP4) inhibitor sitagliptin reduced blood glucose levels, increased GLP1 levels andprevented T2DM-induced DiaCM in mice by shifting the energy substrate utilization in the heart from fatty acids towardsglucose [41,42]. Recently, sodium-glucose cotransporter type 2(SGLT-2) inhibitors, novel hypoglycemic agents that increaseurinary Na and glucose excretion, were introduced to DMand DiaCM research and have come into the spotlight. In addition to the beneficial effects of SGLT-2 inhibitors on glucoselowering or natriuretic action, several potential cardioprotective mechanisms of SGLT-2 inhibitors have been reported[5,13,43]. A number of studies have shown the multiple effectsof SGLT-2 inhibitors on cardiac iron homeostasis, antioxidative stress, anti-inflammation, RAAS activity, antifibrosis, andGlcNAcylation, as well as mitochondrial function in the heart[43-47]. Excessive O-GlcNAcylation following chronic activation of the hexosamine biosynthetic pathway is associated withposttranslational modifications in the diabetic heart. OGlcNAcylation impairs cardiac mitochondrial function, Ca2 homeostasis, and ER stress in DM. A previous study showedthat dapagliflozin prevented DiaCM by reducing the levels ofO-GlcNAcylated protein in diabetic mice. These results demonstrated that O-GlcNAcylated levels of FOXO1 reduced bySGLT-2 inhibitors contributed to attenuation of DiaCM andimprovement in heart function [43,46].Oxidative stressExcess generation of reactive oxygen species (ROS) or reactivenitrogen species (RNS) is considered to be a central mechanism for diabetes-associated inflammation and remodeling inthe heart [13,48,49] and contributes to oxidative stress duringboth the early and late stages of DiaCM [50,51]. Defects in theantioxidant defense system further increase oxidative stressduring the later stages of DiaCM [50,51]. Superoxide dismutase (SOD) has an important role in preventing cardiacdamage in the setting of DM. Injection of the SOD mimic mitochondria-targeted mitochondrial triphenylphosphoniumchloride (mito-TEMPO) prevented the hyperglycemia-induced increase in superoxide generation, reduced myocardialhypertrophy and improved myocardial function in STZ-inDiabetes Metab J 2021;45:129-145https://e-dmj.org

Animal models for diabetic cardiomyopathyduced T1DM mice and db/db T2DM mice compared with vehicle treatment [52].The transcription factor nuclear factor erythroid 2-relatedfactor 2 (NRF2) is an essential regulator of the antioxidant response with an important role in preventing diabetes-inducedoxidative stress and cell death. Isolated cardiomyocytes fromNrf2 knockout (KO) mice were more susceptible to high glucose-induced cell death than wild-type (WT) cells [13,53].Furthermore, NRF2-deficient mice were more susceptible todiabetes-induced or angiotensin (Ang) II-induced cardiomyopathy than WT mice, whereas cardiomyocyte-specific overexpression of Nrf2 conferred resistance to Ang II-induced cardiomyopathy [54,55]. Naturally occurring activators of NRF2have been shown to ameliorate diabetes-induced cardiac complications. Sulforaphane is an organosulfur compound derivedfrom cruciferous vegetables such as cabbage, Brussels sprouts,and broccoli that has been shown to upregulate the expressionof numerous genes encoding antioxidant proteins by activatingNRF2 signaling [13,56]. The cardioprotective benefits of sulforaphane in attenuating fibrosis, oxidative damage, inflammation, hypertrophy, and cardiac dysfunction have been demonstrated in both T1DM and T2DM mouse models and in miceexposed to Ang II [54,55,57,58]. Administration of the antioxidant N-acetylcysteine (NAC) for 5 weeks to rat and mousemodels of STZ-induced T1DM normalized the levels of oxidative stress and subsequently prevented the development of DiaCM [59,60]. Interestingly, the earlier the NAC treatment protocol was initiated after induction of diabetes with STZ duringthe 12-week experiment, the greater the protection against DiaCM [60], suggesting that early damage mediated by increasedoxidative stress has a more important role in the developmentof DiaCM. In diabetic rats, NAC treatment attenuated cardiacdysfunction and damage after myocardial ischemia–reperfusion injury [61,62].Myocardial fibrosis and hypertrophySystemic inflammation, hyperglycemia, and dyslipidemia associated with DM lead to the development of cardiac fibrosisand hypertrophy, which increase myocardial stiffness and result in LV diastolic and systolic dysfunction [13].In DiaCM, increased collagen accumulation is observed inperivascular loci, intermyofiber spaces, and replacement fibrosis [14]. Thus, cardiac fibrosis increased in some animal models of both T1DM [14,63-66] and T2DM [67,68]. Under diabetic conditions, advanced glycation end products created byhttps://e-dmj.orgDiabetes Metab J 2021;45:129-145the exposure of proteins and lipids to high glucose levels crosslink extracellular matrix (ECM) proteins, impair ECM degradation by matrix metalloproteinases and increase cardiac stiffness, which together manifest as early LV diastolic dysfunction[13,69,70]. Genetically obese mice exhibited severe diastolicdysfunction, as evidenced by decreasing the ratio of the early(E) to late (A) (E/A) velocities in db/db and ob/ob mice [21,71,72]. Contractile properties were still slightly affected in ob/obmice [75], while db/db mice displayed reduced fractionalshortening and velocity of circumferential fiber shortening at12 weeks of age [21,72].Epicardial and endothelial cells can also contribute to thedevelopment of cardiac fibrosis through epithelial-to-mesenchymal or endothelial-to-mesenchymal transition to myofibroblasts [13,73-75].The antifibrotic agent cinnamoyl anthranilate reduced collagen production stimulated by transforming growth factor β(TGF-β) signaling in cultured renal mesangial cells [76]. Administration of FT23 and FT011, which are derivatives of cinnamoyl anthranilate, attenuated cardiac structural and functional abnormalities in an animal model of DiaCM [77,78].Inflammation and cytokinesIn the diabetic heart, chemokines, cytokines, and exosomes secreted by inflammatory cells contribute to the development ofcardiomyocyte hypertrophy and ECM remodeling. Severalmyocardial processes are activated by a number of proinflammatory factors, dyslipidemia, hyperglycemia, and elevated AngII levels that are upregulated in the setting of DM [13]. Together, these factors promote the infiltration and accumulation ofproinflammatory lymphocytes and macrophages into the lesion site. These inflammatory cells secrete cytokines such asTGF-β, interleukin (IL)-1β, tumor necrosis factor (TNF), IL-6,and interferon-γ that can cause or exacerbate myocardial injury, contributing to further adverse cardiac remodeling [79,80].Mice with STZ-induced T1DM have higher T cell infiltration into the myocardium, which is associated with increasedmyocardial fibrosis and LV dysfunction, than control mice[81]. Inhibition of T cell trafficking in diabetic mice preventedmyocardial fibrosis and cardiac dysfunction [82,83].Toll-like receptor 4 (TLR4) is expressed in cardiomyocytes,inflammatory cells, and cardiac fibroblasts in both normal andfailing hearts [13]. The role of TLR4-mediated inflammatorysignaling in the development of DiaCM has been reported inanimal models of T1DM and T2DM [84,85]. Inflammatory133

Lee WS, et al.factors, including nuclear factor-κB and TNF, and protein kinases, such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), can directly lead to cardiomyocyte hypertrophy and can advance myocardial fibrosis[86,87]. Activation of the NLR family pyrin domain containing3 (NLRP3) inflammasome, a regulator of cell death and inflammation [88], has been associated with cardiac inflammation, fibrosis, and cell death triggered by HFD and STZ administration in a rat model of T2DM [89]. These effects were attenuated by microRNA (miRNA)-mediated Nlrp3 silencing[89] or by pharmacological suppression of NLRP3 inflammasome activation [90].Suppression of TLR4 signaling with triptolide or matrineimproved cardiac LV function and reduced collagen accumulation in rat models of DiaCM [91,92]. Long-term blockade ofTLR4 with the TLR4 inhibitor TAK-242 (also known as CLI095) was associated with a slight improvement in diabetes-induced erectile dysfunction in rats compared with no treatment, mediated by an increase in cyclic guanosine monophosphate levels and the attenuation of oxidative stress in penile tissue [93]. Numerous small-molecule inhibitors of the NLRP3inflammasome have evolved in the past several years. Theorally active NLRP3 inhibitor 16673-34-0 prevented Westerndiet-induced systolic and diastolic LV dysfunction in obesemice [94].Cardiomyocyte damage and apoptosisApoptosis is an extremely controlled mechanism of programmed cell death and seems to be the principal form of celldeath in DiaCM, compared with lower rates due to necrosis[95,96].In T1DM animals, both increased death receptor signalingand mitochondria-dependent proapoptotic signaling led to elevated apoptosis in DiaCM, and antioxidant treatment diminished both of these signaling pathways and apoptosis, suggesting an essential role of increased ROS in apoptosis inductionin DiaCM [95,97]. A recent study also proposed that dissociation of B-cell lymphoma 2 (Bcl-2) protein from beclin-1 byrestoration of impaired AMP-dependent protein kinase(AMPK) activity may decrease apoptosis in DiaCM by restoring autophagy, supporting the suggestion that an interplay between apoptosis and autophagy may be important in DiaCM[98]. Furthermore, ER stress may encourage apoptosis in DiaCM by activating JNK signaling and apoptosis via the extrinsic and intrinsic pathways or by increasing protein kinase134RNA-like ER kinase (PERK)-C/EBP homologous protein(CHOP) signaling, which may provoke apoptosis by switchingexpression towards proapoptotic Bcl-2 proteins [99].Impaired CA2 handlingIn DM, the process of cardiac calcium cycling (Ca2 entry, intracellular Ca2 concentration, and Ca2 efflux) is modified inboth humans and animal models, contributing to impairedcardiac contraction and relaxation. Decreased Ca2 entry is theconsequence of both altered voltage dependence of the L-typecalcium channel (LTCC) and reduced expression [95]. Impaired intracellular Ca2 cycling consists of reductions in theamplitude of Ca2 and in the systolic rate of the Ca2 rise andfall [100,101]. Prolonged rates of Ca2 decay may arise fromimpaired sarco/endoplasmic reticulum Ca2 -ATPase 2a (SERCA2a) activity during the diastolic period, which may cause adecrease in sarcoplasmic reticulum (SR) Ca2 storage of up to50% and, thus, can lead to diastolic dysfunction and impairedrelaxation [102].In models of T2DM, contractile dysfunction may be drivenby a significant decrease in the Ca2 transient due to reducedCa2 influx as a consequence of decreased LTCC expression, bydecreased SR Ca2 content due to increased phospholambanexpression and decreased SERCA2a expression, and by the diminished activity and content of ryanodine receptor (RyR)[95,103]. In addition, hyperglycemia may lead to the OGlcNAcylation of Ca2 /calmodulin-dependent protein kinaseII (CaMKII), which may accelerate diastolic SR Ca2 leakagevia RyRs, leading to SR Ca2 depletion [104].A potent late Na current inhibitor, ranolazine, might normalize altered intracellular Ca2 levels in cardiomyocytes dueto the close relationship between Ca2 and Na coupling handled by the Na /Ca2 exchanger [5,105]. Ranolazine improvedseveral hemodynamic parameters but not cardiac relaxationvariables. This result showed that a single treatment using ranolazine is probably not sufficient to influence myocardialstructure and cardiac function [5,105].Renin-angiotensin-aldosterone system activationCurrent evidence from animal experiments and human patients has identified a critical role for RAAS in DiaCM [5]. Cytoplasmic Ang II enhances cell growth in animal models. AngII has a definite influence on cell signaling, resulting in cardiomyocyte hypertrophy and proliferation of cardiac fibroblasts[106]. Other factors, such as inflammation, oxidative stress,Diabetes Metab J 2021;45:129-145https://e-dmj.org

Animal models for diabetic cardiomyopathyand aldosterone, may potentiate the harmful effects of Ang IIon the heart that lead to myocardial damage in DM [107].Moreover, the enhanced activation of Ang II and mineralocorticoid receptor signaling might promote insulin resistance byinitiating the mammalian target of rapamycin (mTOR)-S6 kinase 1 signal transduction pathway [5,108].Recently, renin inhibitors (aliskiren), angiotensin II receptorblockers (ARBs), and angiotensin converting enzyme inhibitors (ACEis) were shown to be protective medications againstDiaCM in rat models [5,109]. ACEis and ARBs were also useful agents in both human and animal models of DiaCM [110,111]. The favorable effect of β-adrenoreceptor blockers wasalso demonstrated in experimental models of DiaCM [112].Mitochondrial dysfunctionMitochondrial dysfunction is a well-known feature of DiaCMin both animal and human DM. Mitochondrial dysfunctionrefers to abnormal mitochondrial ultrastructure, increased mitochondrial oxidative stress, impaired activity of Ca2 -sensitivedehydrogenases and F0F1-ATPase, increased sensitivity forCa2 -induced opening of the mitochondrial permeability transition pore, transcriptional and translational downregulation ofoxidative phosphorylation (OXPHOS) subunits, and impairedmitochondrial respiratory capacity and coupling [95,113].In humans, several studies have demonstrated mitochondrial dysfunction in the atrium and atrial appendages of DM patients [114-116], with impaired respiration rates and electrontransport chain complex activities in patients with DM. In adiabetic mouse model, as early as 1985, an impairment in state3 respiration of isolated cardiac mitochondria was observed[117]. Since then, mitochondrial dysfunctions have been reported in numerous diabetic rodent models [118]. In terms ofT1DM models, STZ-treated rats showed reduced antioxidantglutathione, increased ROS production, and ultimately loss ofmitochondrial membrane potential [119]. OVE26 mice alsodisplayed a reduction in glutathione, altered mitochondrialfunction, and an increase in mitochondrial biogenesis [120].Akita mice revealed an increased volume of mitochondria withreduced crista densities and respiratory defects [121]. InT2DM models, db/db mice displayed increases in O2 consumption, lipid peroxidation, and mitochondrial ROS generation [122]. Otsuka Long-Evans Tokushima fatty (OLETF) andob/ob mouse models maintained unchanged levels of uncoupled proteins despite mitochondrial dysfunction [123,124].Zucker diabetic fatty (ZDF) rats showed increased lipid peroxhttps://e-dmj.orgDiabetes Metab J 2021;45:129-145idation and mitochondrial ROS production rates with elevatedantioxidant levels [125,126]. Goto-Kakizaki (GK) and OLETFrats also revealed higher lipid peroxidation and mitochondrialROS production [125,127,128]. The activity of Sirtuin 3(SIRT3), a major regulator of intramitochondrial protein acetylation and NAD -dependent mitochondrial deacetylase, maybe reduced in the diabetic heart, causing ROS deposition dueto increased acetylation and, thus, suppression of manganesesuperoxide dismutase (MnSOD) [95,129]. Furthermore,SIRT3 deficiency seems to exacerbate suppression of mitophagy and autophagy in the diabetic heart, whereas SIRT3 overexpression promoted mitophagy and autophagy, attenuated cardiomyocyte apoptosis and diminished mitochondrial defects[130].MicroRNAsIn DiaCM, dysregulations of 316 out of 1,008 total miRNAswere discovered, and pathway analysis demonstrated that several miRNAs are involved in cardiac hypertrophy, oxidativestress, apoptosis, and autophagy [95,131].Adenovirus-mediated rescue of the myocardial proviral integration site for Moloney murine leukemia virus-1 (Pim-1)expression in vivo improved systolic and diastolic function, attenuated apoptosis and fibrosis, attenuated ventricular dilation,and restored SERCA2a content in DiaCM [132,133]. The expression of miR-133 is decreased in the STZ-induced diabeticmice, and miR-133 has direct inhibitory effects on collagen deposition by deteriorating connective tissue growth factor expression, indicating that increased miR-133 concentrationsmay attenuate myocardial fibrosis in DiaCM [134]. Myocardialexpression of miR-451 is distinctly increased in mice fed aHFD, and cardiomyocyte-specific deletion of miR-451 decreases ceramide deposition, cardiac hypertrophy, and myocardial fibrosis in this mouse model. Diminution of hypertrophy may come from restoration of attenuated AMPK activity,which may normalize increased mTOR phosphorylation andthus restrict HFD-induced cardiomyocyte growth [135]. Upregu

HFD/HSD [14,21] Mice Diet-induced Feeding 1 wk Obesity, high TG HFD low dose STZ [14,21] Mice Diet & Pharmacological Feeding injection β-Cell 2-10 wk Obesity, IR ob/ob [14,21] Mice Transgenic Deficiency Leptin 8-15 wk Obesity, IR, high TG, FFA db/db [14,21] Mice Transgenic Nonfunctioning Leptin receptor 4-8 wk Obesity, IR, high TG, FFA